Coxiella burnetii, the etiologic agent of Q fever, is an obligate intracellular bacterium that replicates within an apparently unmodified phagolysosome. Among intracellular pathogens, the organisms are novel in their location of replication, extreme stability to stress, and persistence in the environment. Bacterial replication is controlled primarily by activated macrophage / monocyte and PMN killing mechanisms stimulated by a cell-mediated response, but the exact nature of these mechanisms is undefined. The author proposes that survival mechanisms are the principle virulence determinants of C. burnetii. Four strategies will be evaluated for their contribution to survival. First, the applicant plans to define the nature and role of the C. burnetii lifecycle in survival. The working hypothesis is that separable, morphological variants represent stages of cell differentiation with specific roles in intracellular and extracellular survival. Based upon earlier studies and data in the preliminary studies, two major variant forms (large cell variants and small cell variants) differentially express proteins that support a model of metabolically most active dividing cells and stationary forms, respectively. Second, he will characterize the requirement for and acquisition systems used to obtain and regulate iron. The working hypothesis is that C. burnetii must accommodate conditions of limiting and high iron levels to survive in the phagolysosome. Data presented in preliminary studies demonstrate a C. burnetii ferric uptake regulator (fur) gene. The author has begun to define proteins involved in iron acquisition using a ferric uptake regulator titration assay (FURTA). Third, he plans to characterize the role of anti-oxidant gene products in survival. The working hypothesis is that C. burnetii express enzymes that detoxify oxygen radicals outside of their cytoplasm and respond to oxidative stress by repairing DNA damage caused by oxygen radicals. Data presented in preliminary studies is expected to pave the way for characterizing catalase, periplasmic superoxide dismutase (Cu/Zn SOD) and RecA. He will test molecular Koch's postulate for the requirement of RecA by creating a transdominant negative mutant through genetic transformation. Finally, he will characterize the role that acid phosphatase plays in intracellular survival. The working hypothesis is that C. burnetii expresses acid phosphatase which phosphorylates phagolysosomal proteins and reduces their antibacterial activity. The author predicts that his approach will define major survival and virulence determinants. The results are expected to be significant, as C. burnetii is a unique and valuable comparative model for intracellular parasitism. In addition, C. burnetii is a common although infrequently diagnosed agent that causes fever of unknown origin and a potential bioterrorist agent. Identification of primary virulence determinants may provide focus for therapeutic, diagnostic and vaccine development.